Induced pluripotent stem cells (iPSCs) from human patients represent a novel source of cells for regenerative medicine. Because iPSCs can be derived from the tissues of recipient patients, they address two major problems in the field of human embryonic stem cells (hESCs), namely immune rejection and the ethical concerns regarding the destruction of embryos. Understanding the epigenomic states of reprogramming cells will give pivotal insights into the efficiency, safety, and efficacy of iPSCs. To date, the efficiency of iPSC reprogramming is very low at 0.001% to 1%. As iPSC lines are clonal, and derived from a single patient, all cells in differing stages of the transition to iPSC share a common genome. Thus, epigenomic changes underly the reprogramming process. Yet, to date, there have been virtually no studies to explore the epigenomics of the actual reprogramming process itself due to technological difficulties. Novel methods described herein will allow the study of this interval for the first time. First, by use of "biopsies" with a micropipette, cells of colonies whose fates can be observed prospectively can be procured. Second, an innovative method of amplifying nucleic acids of small numbers of cells allows second generation sequencing technology to be employed. By these methods, the epigenomics changes of colonies can be tracked as they reprogram. The molecular features which are associated with abortive and successful de-differentiation can be defined. Two other topics will be covered in this program project: tissue of origin and aging. Studies to date have shown different tissues de-differentiate at varying efficiencies, and cells of younger subjects are more easily reprogrammed. By understanding the epigenomic landscapes of different tissues and ages and tracking changes during reprogramming, we hope to identify even more features associated with successful and abortive de-differentiation. PUBLIC HEALTH RELEVANCE: Discovering features associated with successful and abortive de-differentiation will form the basis by which reprogramming can be improved, both in efficiency and differentiation potential, by molecular manipulations. This development should bring iPSCs closer for their use in patients for regenerative medicine.